Bottom Line:
Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction.Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death.The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

ABSTRACTPolo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

Figure 1: Minimization of PBIP1 p-T78 peptide that binds to Plk1. (a–c) Various lengths of N-terminal Cys-(CH2)6-fused T78 peptides were cross-linked to the beads (a) and then tested for their ability to bind to Plk1. The Ser residue (blue) in (a, right) indicates the invariable S77 residue critical for PBD binding. For comparison, a shortened form of the synthetic peptide optimized for Plk1 PBD binding (MQSpTPL)13 was included. (d) A 6-mer T78 peptide (LHSpTAI) analogous to the synthetic optimal peptide (MQSpTPL) was tested for Plk1 binding. Numbers indicate the efficiency of Plk1 precipitation by each peptide relative to the Plk1 signal in the input.

Mentions:
PBIP1/MLF1IP/KLIP1/CENP-50/CENP-U (PBIP1 hereafter) was isolated as a PBD-interacting protein critical for centromeric localization of Plk114 and proper chromosome segregation14–17. Further investigation on the Plk1-PBIP1 interaction shows that the PBD of Plk1 binds to the T78 region of PBIP1 in a phospho-dependent manner14. To better understand the binding nature of this interaction, we synthesized various p-T78 peptides for in vitro binding analyses. A bead-immobilized 10-mer or 14-mer p-T78 peptide, but not the respective non-phospho forms, precipitated Plk1 from mitotic HeLa cells as the major binding protein (Supplementary Fig. 1a). To determine a minimal sequence of the T78 motif required for the interaction, we carried out systematic deletions from the 10-mer p-T78 peptide (PLHSpTAIYAD) and tested the ability of various resulting peptides (Supplementary Table 1) to bind to Plk1. To our surprise, removal of all the amino acid residues C-terminal to the p-T78 residue did not diminish the level of Plk1 binding (Fig. 1a, left), suggesting that the residues after p-T78 are dispensable for PBD binding. Further N-terminal deletion analyses of PLHSpT showed that LHSpT lacking the N-terminal Pro possessed a greatly diminished binding affinity to Plk1, while HSpT lacking both the Pro and Leu residues did not exhibit any detectable level of binding (Fig. 1a, right). These results suggest that PLHSpT binds to Plk1 with high affinity and that, besides the SpT dipeptide, the N-terminal Pro-Leu motif is required to provide an additional level of affinity to the PBD.

Figure 1: Minimization of PBIP1 p-T78 peptide that binds to Plk1. (a–c) Various lengths of N-terminal Cys-(CH2)6-fused T78 peptides were cross-linked to the beads (a) and then tested for their ability to bind to Plk1. The Ser residue (blue) in (a, right) indicates the invariable S77 residue critical for PBD binding. For comparison, a shortened form of the synthetic peptide optimized for Plk1 PBD binding (MQSpTPL)13 was included. (d) A 6-mer T78 peptide (LHSpTAI) analogous to the synthetic optimal peptide (MQSpTPL) was tested for Plk1 binding. Numbers indicate the efficiency of Plk1 precipitation by each peptide relative to the Plk1 signal in the input.

Mentions:
PBIP1/MLF1IP/KLIP1/CENP-50/CENP-U (PBIP1 hereafter) was isolated as a PBD-interacting protein critical for centromeric localization of Plk114 and proper chromosome segregation14–17. Further investigation on the Plk1-PBIP1 interaction shows that the PBD of Plk1 binds to the T78 region of PBIP1 in a phospho-dependent manner14. To better understand the binding nature of this interaction, we synthesized various p-T78 peptides for in vitro binding analyses. A bead-immobilized 10-mer or 14-mer p-T78 peptide, but not the respective non-phospho forms, precipitated Plk1 from mitotic HeLa cells as the major binding protein (Supplementary Fig. 1a). To determine a minimal sequence of the T78 motif required for the interaction, we carried out systematic deletions from the 10-mer p-T78 peptide (PLHSpTAIYAD) and tested the ability of various resulting peptides (Supplementary Table 1) to bind to Plk1. To our surprise, removal of all the amino acid residues C-terminal to the p-T78 residue did not diminish the level of Plk1 binding (Fig. 1a, left), suggesting that the residues after p-T78 are dispensable for PBD binding. Further N-terminal deletion analyses of PLHSpT showed that LHSpT lacking the N-terminal Pro possessed a greatly diminished binding affinity to Plk1, while HSpT lacking both the Pro and Leu residues did not exhibit any detectable level of binding (Fig. 1a, right). These results suggest that PLHSpT binds to Plk1 with high affinity and that, besides the SpT dipeptide, the N-terminal Pro-Leu motif is required to provide an additional level of affinity to the PBD.

Bottom Line:
Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction.Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death.The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.

ABSTRACTPolo-like kinase-1 (Plk1) has a pivotal role in cell proliferation and is considered a potential target for anticancer therapy. The noncatalytic polo-box domain (PBD) of Plk1 forms a phosphoepitope binding module for protein-protein interaction. Here, we report the identification of minimal phosphopeptides that specifically interact with the PBD of human PLK1, but not those of the closely related PLK2 and PLK3. Comparative binding studies and analyses of crystal structures of the PLK1 PBD in complex with the minimal phosphopeptides revealed that the C-terminal SpT dipeptide functions as a high-affinity anchor, whereas the N-terminal residues are crucial for providing specificity and affinity to the interaction. Inhibition of the PLK1 PBD by phosphothreonine mimetic peptides was sufficient to induce mitotic arrest and apoptotic cell death. The mode of interaction between the minimal peptide and PBD may provide a template for designing therapeutic agents that target PLK1.